Natural food that meet antibiotics resistance challenge: In vitro synergistic antimicrobial activity of Azadirachta indica, Terminalia chebula, piper nigrum

and photoactivated cow urine

Objective: Evolution, inappropriate and over use of antibiotics is responsible for antibiotics and drugs resistance among the microbes. Drug resistance microbes can increase the chances of death, complications of illness, requires prolonged treatment and more hospital stays. This situation makes immune-compromised patients more prone to nosocomial infections. Mother Nature has been inexhaustible and infinite source for medicinal compounds. Asian communities are using spices, cow urine and medicinal plants extracts in their diets and naturopathy. Methods: Antimicrobial activities of photo activated Cow Urine, Azadirachta indica, Terminalia chebula, and Piper nigrum were tested on the basis of Minimum Inhibitory Concentration which was tested singularly as well as with all possible combinations by agar diffusion and broth dilution methods against Streptomyces aureofaciens (MTCC 325), E.coli (MTCC 448), Streptococcus mutans (MTCC 497), Pseudomonas aeruginosa (MTCC 7093) Candida albicans (MTCC183), Candida paraopsilosis (MTCC 1965), Candida tropicalis (MTCC 184) and Candida glabrata (MTCC 3019).Results: Motivating results were obtained. Azadirachta indica, Piper nigrum, terminalia chebula and Cow urine has shown remarkable antimicrobial effects. On an average MIC for cow urine, Azadirachta indica Piper nigrum, terminalia chebula was 200l, 70-100l, 100l and 90l respectively. T.chebula showed superb antimicrobial activity against C. propsiolosis, S. aureofaciens, S. mutans and C. albicans. It also has moderate antimicrobial activity against P. aeruginosa, E.coli, C. tropicalis and C. glabrata. A. indica was antimicrobial against S. aerufaciens, C. albicans, E. coli, P. aeruginosa and C. glabrata. Cow urine was antimicrobial agains E.coli, C. albicans and S. aeruginosa. Piper nigrum was antimicrobial against S. aerufaciens, C.tropicalis and C. propsiolosis. Remarkable synergistic effects were observed where cow urine and T. chebula, A. indica and cow urine, T. chebula and Piper nigrum, cow urine and Piper nigrum, A. indica and Piper nigrum, A. indica and T. chebula, and also when used in a combination of three and four and the antimicrobicity against all microbes got enhanced. Conclusion: The results from this study suggest the effects of these natural agents against pathogenic microbes were impressive but depend upon the combination used and tested against which microbes. Overall the synergistic and singular use inhibited the growth of microbes. Results from this study reconfirms and strengthen our position to naturally tackle the challenge of disease caused by pathogenic microbes and can be helpful in finding a solution to outdating of antibiotics and decreases our dependence on them.

Key words: Antibiotics, antimicrobial resistance, pathogens, antimicrobial.

Received : 24 Mar 2012 Accepted : 12 Apr 2012 Published:10 Jun 2012

INTRODUCTION new dawn was seen when antibiotics were Adiscovered and we were assured to get rid of

deadly disease caused by microbes. It was Fleming who [1]discovered Penicillin in year 1928 and Penicillin became open

to the market in year 1944. Within one year of launch we had our first antibiotic resistance strain Penicillin Resistance S. aureus (PRSA). To tackle the challenge, Methicillin a new drug effective against PRSA was launched in the market in year 1959 but we had

[2]Methicillin resistance S. aureus (MRSA) mocking at us in 1960 . Antibiotics have been losing their edge in fight against microbes

[3] .Many antibiotic resistance microbes like Vancomycin resistant Enterococcus (VRE), PRSA, MRSA, Quilone and Ciprofloxacin resistance P. Aeruginosa (QCPRA) pose a challenge to our well being. Many food borne pathogens such as E.coli, Salmonella and Campylobacter responsible for diarrhoea and gastroenteritis

has resistance towards antibiotics. Sexually transmitted bacteria responsible for gonorrhoea, Penicillin resistant Streptococci causative agent for pneumonia, microbes responsible for tuberculosis, influenza, HIV and malaria all have become

4antibiotics resistant. Indiscriminate use of antibiotics has helped microbes to develop resistance and cause adverse effects like hypersensitivity, allergic reactions and immune system

[5, 6]suppression to the user . Multiple antibiotic resistant microbes are held responsible for nosocomial infections and complicate treatment for conditions like cancer, joint replacement and heart

[7]surgery . In the developing countries drugs are not only [27]expensive but also cause various disorders during treatment .

Resistance among microbes is the outcome of production of new enzymes, which help them to resist the drug

[5]qualitatively and quantitatively . A need has been felt to develop new strategies to meet challenges posed by multidrug resistance microbes. Nature has always been the foremost source of medicinal and valuable agents. Old civilizations like Indian, Chinese, Yunani, Egyptian and African have relied on herbal form

[8]of healthcare . Herbs and spices have medicinal health

Olojo E.A.A., Awoniran R. ,Olurin K.B.

1Department of Biotechnology,Motilal Nehru National Institute of Technology,Allahabad-211004, Uttar Pradesh, India.2Department of Biotechnology,University Institute of Engineering And Technology, Kurukshetra University, Kurukshetra-136119,

122

Abstract

Address for correspondence*Siddharth VatsDepartment of Biotechnology,Motilal Nehru National Institute of Technology, India.Email :vatssidd@gmail.com

Asian Journal of Pharmaceutical and Biological Research Original Article

e ISSN: 2231-2218 2012 Asian J Pharm Biol Res

1 2 1Siddharth Vats* ,Rajesh Kumar ,Sangeeta Negi

Asian J Pharm Biol Res |Apr-Jun 2012 | Vol-2 | Issue-2

benefiting properties with addition to their flavour and fragrance 30qualities. India is one of seven hot spots in terms of biodiversity

of flora and fauna. The land has provided habitat to rich medicinal plants and 30 percent of the world's cattle. Out of which 7500 plants species with proven medicinal value and 26 different breeds of cow the walking dispensary are indigenous

[9, 10]to India .

Numerous studies has been published on the antimicrobial activities of plants extracts against different types

[28, 29] of microbes but results reported out of those studies are very difficult to compare directly and have never touched use of spices and photo-activated cow urine synergistically against bacterial, fungal and yeast strains. In the present study we have focused on the synergistic effect of Azadirachta indica, Terminalia chebula, Piper nigrum and cow urine.

In India, Azadirachta indica called as "Panacea for all diseases. Neem tree was described as Azadirachta indica by De Jussieu in 1830. He was also credited for taxonomic position of

[11]Neem . Sanskrit name of Neem is Arishtha means reliever of sickness. Its value is recognized by U.S. National Academy of Science, considering it as a tree for solving global problems. It was Siddique who in 1942 isolated Nimbin the first compound isolated from Neem oil. Till now more than 135 compounds have

[11]been isolated from Neem . Countries where professional dental care is limited Neem is really a potion for dental care. Terminalia chebula a native plant of South Asia, China and Vietnam produces small sized, ribbed nut like fruits which is profoundly used in pickles, spices and medicines. It has been said by Ayurvedic geniuses that sometimes mother can harm a child when she is angry but Terminalia chebula fruits can't harm when it is taken as medicine. So it can be used even to the immune-compromised patients. It has very high antioxidation activity too [12].

Piper nigrum has found its use in the treatment of cancer by stimulating melanocytes proliferation and dendritic formation and so was considered as the potent weapon against disorders like

[13]vitiligo . Trikatu which is a prepared from different kinds of piper and ginger has shown to posses diverse biological activity

[14]in mammalian systems . Black pepper aids in digestion, fight tooth decay, powerful antioxidant, and have antimicrobial

[15]activity against pathogens . Cow urine contains volatile salts of potassium, chloride, calcium of which profoundly benefits human body and acts like a magical potion for the treatment of cancer, asthma, chronic renal failure, Eczema, Psoriasis, acne

[16, 17, 18, 19]vulgaris, scabies and allergies . Photo activation of Cow urine produces metabolically active secondary nitrogenous compounds, also a decrease in pH makes cow urine more

[5, 20]antimicrobial . Sulfinol, Ketones, formaldehyde formation [21]during photo-activation made it more antimicrobial . Our

surrounding is full of microbes. Temperature and nutrients present in the body provide a five star welcome to them. And the use of Azadirachta indica and gou-Muttra as immune stimulants

[22]is well noted .

MATERIALS AND METHODSMicroorganisms

Bacterial strains used were Streptomyces aureofaciens (MTCC 325), E.coli (MTCC 448), Streptococcus mutans

(MTCC 497) and Pseudomonas aeruginosa (MTCC 7093). Fungal strains used were Candida albicans (MTCC183) and yeasts like Candida paraopsilosis (MTCC 1965), Candida tropicalis (MTCC 184), and Candida glabrata (MTCC 3019). These were the pathogens used in the study. All the cultures were obtained in pure form from the culture collection of Institute of Microbial Technology (IMTECH), Chandigarh, India.

Preparation of herbal extracts of Azadirachata indica

Fresh leaves of Azadirachta indica were taken during the monsoon season. Leaves were washed with boiling water to remove microbes and dust present over the surface. Washed leaves were air dried and grinded to juice with the pestle and mortar. For each 10 gm of leaves 25ml of water was used to ease the grinding. Juice was obtained and centrifuged @ 5000 rpm for 10 minutes at 4C. Super natant was taken and extract was considered as 100 % pure.

Collection of Cow urine

Fresh cow urine was taken from Indian breed cow. Cow urine used was photo-activated by keeping in a transparent bottle in sunlight in the daytime and in artificial light at night for 48 hours. And then centrifuged at 10000 rpm for 15 minutes to remove all debris and precipitated material. Super natant was collected and filtered through 0-45 micron filter. Before performing the test against microbes cow urine was tested for its sterility. To check it was free of microbes it was inoculated in broth and also nutrient agar.

Preparation of extracts of Terminalia chebula and Piper nigrum

Dried fruits of plants are taken and crushed in to the powder form. Then 10 gm powder / 25 ml of water are mixed and formed a viscous solution. This juice is then centrifuged for 15 minutes at 7000 rpm. Supernatant was collected for antimicrobial

0activity test. This was stored at 4 C for further use.

Determination of minimum inhibitory concentration

To find the minimum inhibitory concentration agar diffusion method and broth dilution method were followed. For agar diffusion method plates containing Muller Hinton agar (MHA) were made and then spread with each of different strains and well of 12 mm was made at centre. Different volume of extract was added to find at which minimum volume of extracts growth of microbe was inhibited.

Agar diffusion susceptibility test

Agar diffusion method was employed to find antimicrobial activity of the Azadirachta indica, Piper nigrum, Terminalia chebula extracts and Gou Muttra. Mueller Hinton agar (MHA) from himedia was used. Agar plates were made as mentioned in the instructions. Sterilize it by autoclaving at 121 degree Celsius under 15 lb psi (pounds per square inch) pressures. Sterilized agar poured and allowed to solidify in Petri plates in laminar flow. 100 l of 24 hours old microbial culture were then spread on to it by sterile cotton buds. The 0/6 cm diameter of punched hole were loaded with 150 micro liters of extracts and cow urine unary as well as in all possible combination combination and then incubated at 37C for 24 hours. Triplicate tests were made for each experiment. The diameter in mm of inhibition zones around each of the holes was recorded at the end of the incubation time.

123

Asian J Pharm Biol Res |Apr-Jun 2012 | Vol-2 | Issue-2

Broth dilution susceptibility test

In case of broth dilution method, different volume of plants extracts and cow urine were added in the Muller Hinton broth. Microbes were inoculated into these tubes. The tubes were incubated to allow microbial growth for 24 hours. The interpretation of this MIC depends on the organism and antimicrobial pair being tested.

RESULTS AND DISCUSSIONAmong the three plant extracts and cow urine tested, all

has antimicrobial activity. The test was carried out at triplicates and average results were represented in the graphs. The results indicated that MIC for Cow urine was 200 ulfor Azadirachta indica 70-100uland terminalia chebula 90ul respectuively. Equilibrated amount of extract were used in unary, binary, ternary and quaternary combinations.

Figure: 1 The zone of inhibition in the case of P. aeruginosa

Figure: 2 The zone of inhibition in the case of S. mutans

Figure: 3 The zone of inhibition in the case of C. albicans

Figure:4 The zone of inhibition in the case of S aureofaciens

Figure: 6 The zone of inhibition in the case of C. tropicalis

Figure: 7 The zone of inhibiton in the case of C.propsilosis

Figure: 8 The zone of inhibition in the case of E.coli.

124

Figure :9 Synergistic activity of various extracts

In case of P. Aeruginosa the most effective combination was cow urine and Terminalia chebula with a zone of inhibition 40mm. For Candida albicans maximum inhibition was observed for the ternary combination of Azadirachta indica, Terminalia chebula and Piper nigrum with zone of inhibition 37 mm. For S. mutans binary combination of Azadirachta indica and Piper nigrum was found to be inhibitory with a zone of 45 mm. Similarly for S auereofaciens maximum inhibition was observed of 45mm by the application of cow urine and Terminalia chebula. Piper nigrum, Terminalia chebula and cow urine in combination were most effective against the C. glabrata.with a zone of 35mm. For inhibition of C. tropicalis all extracts and cow urine combination was most effective with a zone of 45mm. Growth of C. propsilosis was inhibited by the use of Terminalia chebula with a zone of 35 mm. Combination of Azadirachta indica and Terminalia chebula was inhibitory against E.coli with 25mm zone of inhibition.

From this study it is clear that natural extracts of spices, cow urine and medicinal plants can be used to treat infection caused by microbes. Similar results were obtained in the case of

[5]Azadirachta indica . Neem oil and cow urine unary as well as in synergistic combinations inhibited the growth of many infection

[5]causing microbes . Antibacterial activity was also noted with [23]the use of spice and medicinal plant Cinnamomum . Neem is

the diverse and versatile medicinal plant and is the unique source of various types of compounds. Antimicrobial activity of photo-activated cow urine against certain pathogenic bacterial strains like Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 11778), Lactobacillus acidophilus (ATCC 53103) and

[5].Micrococcus luteus (ATCC 9341), E.Coli (ATCC 25922) It was found that antimicrobial activity is due to presence of volatile organic ingredients and when photo-activated they become more toxic as CO , NH , CH , methanol, propanol and 2 3 4acetone etc. Nitrosamines are also formed in cow urine on

[24].photoactivation

Phenolic extract of T. chebula is also found to have anti [25]cancerous effects and antimicrobial . Antimicrobial agents

[26]from the plants of P. nigrum L. Were extracted and studied .

CONCLUSIONResults reported from this one of kind study confirm that

nature is the best healer. Synergistics effects resulting from the combinations of Terminalia chebula, Piper nigrum, Azadirachta indica and of cow urine have never been documented earlier. Combined effect of natural substances i.e. drug synergism is a novel concept. Azadirachta indica was found to be most effective against S aureofaciens, Terminalia chebula against C. propsilosis, cow urine against C. albicans and Piper nigrum against C. tropicalis. Phytochemicals from T. chebula, Piper nigrum, A. indica and cow urine in different combinations can be of great significance in therapeutic treatments. Antimicrobial agents present in these Indian spices, medicinal plants and cow urine can be effective against plants, human and animal pathogens.

REFERENCES

1. Rolinson GN: Forty years of beta lactam research. J. Antimicro. Chemother., 1998, 41(6): 589-603.

2. Fleming A: On the Antibacterial Action of Cultures of a Penicillium, with Special Reference to their Use in the Isolation of B. influenz. Britis. J. Experimen. Patholo. 1929, 10: 226-236.

3. Davies, J: Inactivation of antibiotics and the dissemination of resistance genes. Science., 1994, 264: 375-382.

4. Haydel SE, Remenih CM, Williams LB: Broad-spectrum in vitro antibacterial activities of clay minerals against antibiotic susceptible and antibiotic-resistant bacterial pathogens. J. Antimicrob. Chemothe. 2008, 61: 353-361.

5. Upadhyay RK, Dwivedi, P, Ahmad S: Antimicrobial activity of photo-activated cow urine against certain pathogenic bacterial strains. Afric. J. Biotechnol. 2010, 9(4): 518-522.

6. Ahmad I, Mehmood Z, Mohammad F: Screening of some Indian medicinal plants for their antimicrobial properties. J. Ethnopharmaco. 1998, 62(2): 183-193.

7. Palmer KL, Daniel A, Hardy C, Silverman J, Gilmore MS: Genetic Basis for Daptomycin Resistance in Enterococci. Antimicrobi. Agent. Chemother. 2011, 55(7): 3345-3356.

125

Asian J Pharm Biol Res |Apr-Jun 2012 | Vol-2 | Issue-2

8. Vats S, Miglani K: Synergistic antimicrobial effect of cow urine and azadirachta indica on infectious microbes. Intern. J. Pharmaceutic. Sci. Resea. 2011, 2(7): 1781-1785.

9. Preethi R, Vimaldevanathan V, Longanathan M: Antimicrobial and Antioxidant Efficacy of Some Medicinal Plants against Food Borne Pathogens Advan. Biolog. Resear. 2010, 4(2): 122-125.

10. Singh RR: Biodiversity of indigenous cattle and its utility: The Indian cow. The Indian Cow J. 2010, 3(1):29-37.

11. Kaushik B, Chattopadhyay I, Banarjee RK, Bandhopadhyay U: Biological activities and medicinal properties of Neem (Azadirachta indica). Curre. Sci. 2002, 82(11): 122-125.

12. Naik GH, Priyadarshini KI, Satav JG, Banavilikar MM, Sohoni DP, Biyani MK, Mohan H: Comparative antioxidant activity of individual herbal components used in Ayurvedic medicine. Phytochemistry 2003, 63(1): 97-104.

13. Soumayanath A, Venlataswamy R, Joshi M, Faas L, Adejuyigbe B, Drake AF, Hider RC, Young AR: Photochemic. Photobiol. 2006, 82: 1541-1548.

14. Johari RK, Zutshi U: An Ayurvedic formulation 'Trikatu' and its constituents. J. Ethnopharmacolo. 1992, 37(2): 85-91.

15. Tashman, L: The Top Ten Health Benefit of Black Pepper. How Adding A Little Pepper Can Improve Digestion And Heart Health. [http://www.suite101.com, 2010].

16. Cow Urine Therapy In Different Types of Diseases. [http://www.cowurine.com]

17. Urine Therapy and Cow Urine. [www.pharmabiz.com] 18. Shaw SL, Mitlochner FM, Jackson W, Depeters EJ, Fadel

JG, Robinson PH, Holzinger R, Goldstein AH: Volatile organic compound emissions from dairy cows and their wastes as measured by protein-transfer-reaction mass spectrometry. Environ. Scien. Technolo. 2007, 14: 1310-1316.

19. Cowurine Medicine. [http://www.gowgram.org/gavya-products/cow-urine-medicine].

20. Hu W, Murphy MR, Constable PD. Dietary cation-anion

difference effects on performance and acid-base status of dairy cows postpartum. J. Dair. Scien. 2007,90:3367-3375.

21. Turi M, Turi E, Koljalg S, Mikelsaar, M: Influence of aqueous extracts of medicinal plants on surface hydrophobicity of Escherichia coli strains of different origin. Acta, Pathologica, Microbiologica. Et Immunologica. Scandinvica, 1997, 105(12): 956-962.

22. Benny KH, Vanisha L: Immunomodulatory and antimicrobial effects of some traditional Chinese medicinal herbs: A review. Curr. Medicin. Chemist. 2004, 11: 1423-1430.

23. Sandigwad BM, Patil, GG. The invitro antibacterial activity of Cinnamomum species. Asian J. Experime. Biolog. Scien. 2010,1(2):434-439.

24. Stephany RW, Schuller PL: The intake of nitrate, nitrite and volatile N-nitrosamines and the occurrence of volatile N-nitrosamines in human urine and veal calves. IARC Scientific Publications.1978, 19: 443-460.

25. Saleem A, Husheem M, Harkonen P, Pihlaja, K: Inhibition of cancer cell growth by crude extract and the phenolics of Terminalia chebula retz. fruit. J. Ethnopharmacology. 2002, 81(3): 327-336.

26. Dorman HJD, Deans SG. Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J. Appl. Microbio. 2000, 88(2):308-316.

27. Chaudhary NMA, Tariq P: Anti-microbial activity of cinnamomum cassia against diverse microbial flora with its nutritional and medicinal impacts. Pak. J. Bot. 2006, 38(1): 169-174.

28. Hara-Kudo Y, Kobayashi A, Sugita-Konishi Y, Kondo K: Antibacterial activity of plants used in cooking for aroma and taste. J. Food Protecti. 2004, 67: 2820-2824.

29. Beuchat LR. In: Dillon, YM, Board RG, (Eds), Natural Antimicrobial Systems and Food Preservation. CAB International 1994, pp. 167-179.

30. Shan B, Cai, YZ, Brooks JD, Corke H: The invitro antibacterial activity of dietary spice and medicinal herb extracts. Intern. J. Food Microbio. 2007, 117: 112-119.

126

Asian J Pharm Biol Res |Apr-Jun 2012 | Vol-2 | Issue-2

Page 1Page 2Page 3Page 4Page 5